Blood contains various lineages of blood cells having biological functions, such as the erythrocytic lineage associated with oxygen delivery, the megakaryocytic lineage generating thrombocytes, the granulocytic lineage associated with prevention of infections, the myeloid lineage such as monocytes and/or macrophages and the lymphocytic lineage responsible for immunity such as T cells and B cells. All these blood cells differentiate and mature from the common origin, hematopoietic stem cells, and are maintained and generated in an individual throughout its life. Hematopoietic stem cells are defined as cells having both pluripotency which allows them to differentiate into functional cells such as lymphocytes, erythrocytes and leukocytes and the ability to regenerate themselves while maintaining the pluripotency (self-renewal).
Previous studies have revealed that hematopoietic stem cells first diverge two ways into the myeloid lineage and the lymphoid lineage, then differentiate into myeloid stem cells (mixed colony forming cells, CFU-GEMM) and into lymphoid stem cells, respectively. Further, myeloid stem cells differentiate into erythrocytes via erythroid burst forming cells (BFU-E) and erythroid colony forming cells (CFU-E), into thrombocytes via megakaryocyte colony forming cells (CFU-MEG), into monocytes, neutrophils and basophils via granulocyte-macrophage colony forming cells (CFU-GM), and into eosinophils via eosinophil colony forming cells (CFU-EO), while lymphoid stem cells differentiate into T cells via T lymphoid progenitor cells and into B cells via B lymphoid progenitor cells. Among them, cells forming multipotential colonies with diameters of at least 1 mm are called HPP-CFU colony forming cells and are known as the least differentiated hematopoietic progenitor cells, along with mixed colony forming cells (CFU-GEMM). These myeloid stem cells and various hematopoietic progenitor cells derived from them are identified by the properties of colonies they form on soft agar, semisolid methylcellulose media or the like in the presence of various cytokines (Non-Patent Document 1).
In recent years, as a curative therapy for a number of intractable diseases such as various blood diseases attributed to hematopoietic dysfunction and immune dysfunction, cancer, immunodeficiency, autoimmune diseases and inborn error of metabolism, autologous or allogeneic transplantation of hematopoietic stem cells have been performed. Quite recently, the effectiveness of transplantation of CD34+ cells including hematopoietic stem cells in treating cerebral infarction, myocardial infarction and obstructive arteriosclerosis was reported (Non-Patent Documents 2, 3, 4 and 5). Attempts to regenerate nerves and muscles through hematopoietic stem cell transplantation are in progress. For example, nerve regeneration in cerebral infarction model mice through angiogenesis caused by transplantation of cord blood-derived CD34+ cells (Non-Patent Document 2) and the possibility of repair of damaged muscles using CD34+ cells were reported (Non-Patent Document 5 and Patent Document 1). Among them, bone marrow transplantation has been used in many cases of treatment and most established as a standard hematopoietic cell transplantation therapy. However, because for bone marrow transplantation, the human leukocyte antigens (HLA) of the bone marrow donor and the transplant recipient have to match closely, there is a problem that bone marrow from donors are in short supply. Besides, the need for at least 4 days of hospitalization and pain, fever and bleeding caused by collection of a large amount of bone marrow are a heavy burden to donors.
In addition to bone marrow, peripheral blood is also used as an alternative source of hematopoietic stem cells nowadays. Hematopoietic stem cells mobilized from the bone marrow to peripheral blood by administration of granulocyte colony stimulating factor (G-CSF) to a human are used for transplantation after enrichment using a blood cell separator. However, donors for peripheral blood hematopoietic stem cell transplantation have to bear a heavy burden of the need for administration of G-CSF for 4 to 6 consecutive days which may cause side effects (such as blood coagulation and spleen hypertrophy). Besides, because the efficiency of the mobilization of hematopoietic stem cells from the bone marrow to peripheral blood by G-CSF varies from donor to donor, hematopoietic stem cells are not obtained sufficiently in some cases.
Just recently, it has been found that cord blood contains the same degree of hematopoietic stem cells as bone marrow and is useful for hematopoietic stem cell transplantation (Non-Patent Document 6). Because cord blood transplantation does not require complete HLA matching and is less likely to cause severe acute graft-versus-host disease (GVHD) than bone marrow and peripheral blood transplantation, cord blood is established as useful and has been used more frequently. However, because cord blood is obtained in a small amount from one donor and does not contain many hematopoietic stem cells, its use is mainly limited to children.
Furthermore, hematopoietic stem cells are also considered as useful cells for gene therapy of fatal genetic diseases with no effective cure, HIV infection, chronic granulomatosis and germ cell tumor. However, in order to transfect hematopoietic stem cells with a retrovirus vector carrying a target gene efficiently, it is necessary to artificially promote the proliferation of hematopoietic stem cells, which are usually in the stationary phase, by recruiting them into the cell cycle. Besides, in order to be successfully transplanted and express a transgene for a long time, the transfected hematopoietic stem cells have to be kept undifferentiated in culture ex vivo. Therefore, gene transfer by an improved cell culture method has been desired for efficient gene transfer and successful transplantation therapy (Non-Patent Document 7).
Meanwhile, hematopoietic progenitor cells are important for initial hematopoietic recovery after bone marrow or cord blood transplantation and are considered as effective, especially, in preventing early posttransplant infections. Therefore, transplantation of an insufficient number of hematopoietic progenitor cells can delay initial hematopoietic recovery and lower the posttransplant survival rate (Non-Patent Document 8).
To solve the above-mentioned problems with hematopoietic stem cell transplantation and gene therapy, a technique for expanding hematopoietic stem cells and/or hematopoietic progenitor cells ex vivo is demanded, and various culture methods have been attempted so far.
Here, hematopoietic stem cells and hematopoietic progenitor cells, which are to be cultured, are explained. It was revealed that in human, hematopoietic stem cells and various hematopoietic progenitor cells derived from them are found in populations of CD34+ cells expressing the CD34 molecule as a cell surface antigen, and hence hematopoietic stem cells can be enriched as a CD34+ cell population (Non-Patent Document 9). Specifically speaking, they are often enriched by mixing a cell population to be separated with a CD34 antibody labeled with magnetic beads and magnetically collecting CD34+ cells (Non-Patent Documents 10 and 11). CD34+ cell populations contain less than 10% of CD34+CD38− cell populations not expressing the CD38 molecule as a cell surface antigen. It has come to be considered that hematopoietic stem cells are more enriched in CD34+CD38− cell populations than in CD34+ cell populations (Non-Patent Documents 12 and 13). In order to determine the proportion of undifferentiated hematopoietic progenitor cells in a cell population, HPP-CFU colony forming cells are usually counted as mentioned above (Non-Patent Document 14). In recent years, it has become possible to experimentally test for the presence of human hematopoietic stem cells which have bone marrow repopulating ability by using NOD/SCID mice obtained by crossing diabetic mice and immunodeficient mice. The cells detected by this assay are called SCID-repopulating cells (SRC) and considered the closest to human hematopoietic stem cells (Non-Patent Document 15).
Conventional techniques for expanding hematopoietic stem cells and/or hematopoietic progenitor cells will also be explained. As mentioned above, since hematopoietic stem cells are more enriched in CD34+ cells, CD34+ cells are mainly used as the starting cells for expansion. Expansion of hematopoietic stem cells and hematopoietic progenitor cells from CD34+ cells in culture in the presence of a cytokine or a growth factor such as stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-6 (IL-6)/soluble IL-6 receptor complex, interleukin-11 (IL-11), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), flk2/flt3 ligand (FL), thrombopoietin (TPO) and erythropoietin or Notch ligand (such as Delta 1) has been reported (Patent Documents 2 and 3 and Non-Patent Documents 8, 14, 16 and 17). Among them, TPO has especially excellent effect on hematopoietic stem cell expansion and used for in most of cases of expansion (Non-Patent Document 18). Hematopoietic stem cells and hematopoietic progenitor cells expand in culture in the presence of such various cytokines and growth factors, but hematopoietic stem cells expand only by several times. Besides, these cytokines and growth factors are all produced as recombinant proteins, it may be difficult to obtain them for expansion stably, in a large amount, at low cost, or quickly.
For ex vivo expansion of hematopoietic stem cells, coculture systems using a different type of cells as feeder cells in the presence of various cytokines were reported. For example, expansion of hematopoietic stem cells in coculture with human bone marrow stromal cells was attempted (Non-Patent Document 19). An attempt to expand CD34+ cells in the presence of TPO, FL and SCF using mouse bone marrow cell line HESS-5 was also reported (Non-Patent Document 20). However, because these coculture systems use foreign cells, there is a risk that cells infected with an unknown pathogen whose existence has not been confirmed may also be transplanted to patients. Furthermore, when stromal cells from a different kind of animal are used, the stromal cells have to be separated completely from CD34+ cells because otherwise there is a risk of causing immune response in the recipient after transplantation.
In addition, ex vivo expansion of hematopoietic stem cells in culture in the presence of various cytokines such as TPO combined with low molecular weight compounds, not just various cytokines only, has been reported. Examples of such low molecular weight compounds include copper chelators, the combination of a histone deacetylase inhibitor and a DNA methylase inhibitor, all-trans retinoic acid, aldehyde dehydrogenase inhibitors, arylhydrocarbon receptor antagonists and the like (Non-Patent Documents 21, 22, 23 and 24 and Patent Document 4). However, addition of any of them is not effective enough since hematopoietic stem cells expand by only several times, or cells have to be cultured for about 3 weeks.
It is known that treatments which promote rapid hematopoietic and immune recovery after transplantation of hematopoietic stem cells are quite effective in eliminating the risk of infections and shortening hospitalization. As such a treatment, posttransplant administration of the hematopoietic cytokine, granulocyte colony stimulating factor (G-CSF), is conducted in clinical settings (Non-Patent Document 25). However, it is effective only for leukocytes, and effective treatments which promote recovery of blood cells of all lineages through expansion of hematopoietic stem cells and/or hematopoietic progenitor cells before transplantation are demanded.